All organisms respond to extreme environmental conditions by either inducing de novo or dramatically increasing the expression of a number of genes that protect the cell from the deleterious effect of intracellular protein denaturation. These genes encode for a family of proteins called HSPs (heat shock proteins) and other molecular chaperones and cytoprotective proteins. Expression of HSPs and other chaperones is induced upon exposure to a variety of stressors including elevated temperature, oxidative stress, alcohol, hyper- and hypoosmotic stress, transition metals, infections, amino acid analogs, etc. (Morimoto, et al., In The Biology of Heat Shock Proteins and Molecular Chaperones, 1994 (New York: Cold Spring Harbor Press), pp. 417-455). HSPs and other chaperones are involved in basic cellular processes under both stress and normal conditions such as correct folding of nascent polypeptides, binding to exposed hydrophobic regions of denatured or abnormal proteins to prevent their aggregation and promote degradation or assembly, and translocation of proteins into membrane-bound organelles in the cell (see, e.g., Ellis, Trends Biochem Sci., 2000, 25: 210-212; Forreiter and Nover, J. Biosci., 1998, 23: 287-302; Hartl and Hayer-Hartl, Science, 2002, 295: 1852-1858; Haslbeck, Cell Mol. Life. Sci., 2002, 59: 1649-1657; Young et al, Trends Biochem. Sci., 2003, 28: 541-547; Soll and Schleiff, Nature Rev. Mol. Cell. Biol., 2004, 5: 198-208). Expression of some HSPs is essential during embryogenesis (Luft, et al., Chaperones, 1999; 4:162-170). HSPs function primarily by stabilizing partially unfolded states. They do not contain specific information for correct folding, but rather prevent unproductive interactions (aggregation) between non-native proteins. This type of molecular chaperones can be distinguished from other heat-induced proteins acting as direct folding catalysts like peptidyl prolyl-cis/trans isomerases (e.g., cyclophilins, FKBPs) or protein disulfide isomerases (Schmid, Curr. Biol., 1995, 5: 993-994; Guidebook to molecular chaperones and protein-folding catalysts, Gething (ed.), Oxford Univ. Press, 1997; Gothel and Marhiel, Cell Mol. Life. Sci., 1999, 55: 423-436; He et al., Plant Physiol., 2004, 134: 1248-1267; Tu and Weissman, J. Cell Biol., 2004, 164: 341-346; Kadokura et al., Annu. Rev. Biochem., 2003, 72: 111-135). Four major aspects in the life cycle of proteins invoke chaperone activities (Guidebook to molecular chaperones and protein-folding catalysts, Gething (ed.), Oxford Univ. Press, 1997): (i) they ensure that nascent polypeptides emerging from the ribosome are kept in a folding competent state until the whole sequence information is available; (ii) since fully folded proteins cannot be translocated through membranes, chaperones are needed to maintain or create a partially unfolded form of proteins destined for the import into mitochondria or plastids (Braun and Schmitz, Planta, 1999, 209: 267-274; Neupert and Brunner, Nat. Rev. Mol. Cell. Biol., 2002, 3: 555-565; Rehling et al., J. mol. Biol., 2003, 326: 639-657; Soll and Schleiff, Nature Rev. Mol. Cell. Biol., 2004, 5: 198-208); (iii) they stabilize damaged proteins generated as a result of chemical or physical stress and thus facilitate renaturation and/or degradation in the recovery period; (iv) they assist and control assembly and disassembly of multiprotein complexes (Lorimer, Plant Physiol., 2001, 125: 38-41; Kim et al., J. Biol. Chem., 2002, 47: 44778-44783). Many data collected during the last few years indicate that members of different Hsp families act together in so-called “chaperone machines” (Bukau and Horwich, Cell, 1998, 92: 351-366; Walter and Buchner, Angew. Chem.-Int. Edit., 2002, 41: 1098-1113; Young et al., Trends Biochem. Sci., 2003, 28: 541-547), and different chaperone complexes may interact to generate a network for protein maturation, assembly and targeting (Frydman, Annu. Rev. Biochem., 2001, 70: 603-647; Johnson and Craig, Cell, 1997, 90: 201-204; Forreiter and Nover, J. Biosci., 1998, 23: 287-302; Lee and Vierling, Plant Physiol., 2000, 122: 189-198). Although many proteins are potential substrates for chaperone machines, e.g. after stress-induced protein damage, most of them (about 80%) fold in a chaperone-independent manner under normal conditions (Netzer and Hartl, Trends Biochem. Sci., 1998, 23: 68-73). It is assumed that proteins at the surface of the ribosomes may help to stabilize nascent polypeptide chains (Frydman, Annu. Rev. Biochem., 2001, 70: 603-647; Hartl and Hayer-Hartl, Science, 2002, 295: 1852-1858).
HSP family proteins are classified into several groups based on their molecular weight and sequence homology between bacteria, plants and animals. Hsp101 (ClpA/B/X in prokaryotes) are involved in ATP-dependent dissociation of protein aggregates. Hsp90 (HtpG in prokaryotes) function as co-regulators of signal transduction complexes. Under stress conditions, HSP90 binds to exposed hydrophobic regions of denatured proteins, while in the absence of stress it participates in fundamental cellular processes such as hormone signaling and cell cycle control (Pearl, et al., Curr. Opin. Struct. Biol. 2000; 10:46-51). Many regulatory proteins, including steroid hormone receptor, cell cycle kinases, and p53 have been identified as HSP90 substrates (Young, et al., J. Cell Biol. 2001; 154:267-273; Pratt, Annu. Rev. Pharmacol. Toxicol. 1997; 37:297-326). HSP90 is ubiquitously expressed and may constitute up to 1-2% of total cellular protein. Mammalian cells express at least two HSP90 isoforms, HSP90α and HSP90β, which are encoded by two separate genes (Pearl, et al., Adv. Protein Chem. 2001; 59:157-186). Hsp70/Hsp40 (DnaK/DnaJ in prokaryotes) are involved in primary stabilization of newly formed proteins via ATP-dependent binding and release (Mayer, et al., Adv. Protein Chem. 2001; 59:1-44), unfolding and refolding of proteins during their transport across membranes (Jensen, et al., Curr. Biol. 1999; 9:R779-R782; Pilon, et al., Cell 1999; 97:679-682; Ryan, et al., Adv. Protein Chem. 2001:59:223-242), and binding to partially denatured, abnormal, or targeted for proteasome degradation proteins (Zylicz, et al., IUBMB. Life 2001; 51:283-287). HSP70 subfamily includes both constitutive and stress-inducible proteins that are closely related and often referred to as Hsc70 and HSP72 respectively. HSP40 is a co-chaperon for HSP70 class proteins, which modulates ATPase activity and substrate binding properties of the latter (Ohtsuka, et al., Int. J. Hyperthermia 2000; 16:231-245). Hsp60/Hsp10 (GroEL/GroES in prokaryotes) function in cytosol and organelles to stabilize unfolded states and assist refolding or degradation in an ATP-dependent manner. Hsp20 are a family of small HSPs, which includes primate HSP27, rodent HSP25, αA-crystallins and αB-crystallins. HSP25/27 is expressed constitutively and expression increases after exposure to heat, transition metal salts, drugs, and oxidants. Small HSPs form high molecular weight oligomeric complexes (e.g., oligomers consisting of 8-40 monomers) that serve as binding sites for stabilization of unfolded proteins until they can be refolded by HSP70/HSP40 and/or Hsp101 system (Van Montfort, et al., Adv. Protein Chem. 2001; 59:105-156; Welsh, et al., Ann. N. Y. Acad. Sci. 1998; 851:28-35).
The induction of HSP gene expression occurs primarily at transcriptional level and is mediated by a family of transcription factors named HSF (Heat Shock Factor). Only one HSF has been identified in yeast, Drosophila, and C. elegans, and three HSFs have been identified in vertebrates (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Morimoto, Genes and Development, 1998, 12: 3788-3796; Nakai, Cell Stress Chaperones, 1999, 4:86-86-93; Nover et al., Cell Stress Chaperones, 2001, 6: 177-189; recently, a fourth, HSF-like open reading frame (ORF) (HsfY) encoded on the Y chromosome was identified in vertebrates—see Tessari et al., Mol. Human. Reprod., 2004, 10: 253-258). In human cells, three HSFs (HSF1, HSF2, and HSF4) have been characterized (Morimoto, et al., Genes Dev. 1998; 12:3788-3796). HSF1 is ubiquitously expressed and plays the principle role in the stress-induced expression of HSPs. It is an apparent functional analog of Drosophila HSF.
In contrast to few well-defined HSFs in other classes of organisms, plants are characterized by a great complexity of the plant HSF family. The complexity of the plant HSF gene family is thought to allow a highly flexible and efficient response to rapid changes in environmental conditions that accompany the stationary lifestyle of plants (Nover et al., Cell Stress Chaperones, 2001, 6: 177-189; Kotak et al., Plant J., 2004, 39: 98-112). Due to their immobility, plants had to adapt to grow and propagate under extreme environmental conditions, including various environmental abiotic stresses such as dehydration/extreme water deficiency, unfavorable high or low temperatures or abrupt temperature shifts, high salinity, heavy metal stress, acid rain, high light intensities, UV-light, grafting, or the bending of shoots or stems in response to wind and/or rain, or biotic stresses such as pathogen attack.
The model plant Arabidopsis thaliana contains 21 HSF genes, as well as several genes encoding HSF-like proteins (The Arabidopsis Genome Initiative 2000, Nature, 408: 796-815). More than 16 HSF genes were found in tomato (Scharf et al., Mol. Cell. Biol., 1998, 18: 2240-2251; Treuter et al., Mol. Gen. Genet., 1993, 240: 113-125; Boscheinen et al., Mol. Gen. Genet., 1997, 255: 322-331; Bharti et al., Plant J., 2000, 22: 355-365; Bharti et al., Plant Cell, 2004, 16: 1521-1535; Doring et al., Plant Cell, 2000, 12: 265-278; Heerklotz et al., Mol. Cell. Biol., 2001, 21: 1759-1768; Mishra et al., Genes Dev., 2002, 16: 1555-1567; Port et al., Plant Physiol., 2004, 135: 1457-1470; see also reviews by Nover et al., Cell Stress Chaperones, 2001, 6: 177-189; Bharti and Nover, Heat stress-induced signalling; in Plant signal transduction: Frontiers in molecular biology, Scheel and Wastemack eds., Oxford Univ. Press, 2002, pp. 74-115), and many other HSF genes were identified in rice, maize and other species (Goff et al., Science, 2002, 296: 92-100; Yu et al. 2002, Science, 2002, 296: 79-92). The number of plant HSFs continues to grow. More than 60 new class A HSFs were identified from expressed sequence tag (EST) databases, including 19 new HSFs in soybean (34 in total) and at least 23 HSFs in rice (Kotak et al., Plant J., 2004, 39: 98-112).
Plant HSF genes are assigned to three different classes (classes A, B and C) according to their unique structural characteristics (Nover et al., Cell Stress Chaperones, 2001, 6: 177-189). Class A HSF proteins comprise the largest group of HSFs with 15 proteins in Arabidopsis. They contain an activation domain at the C-terminus and are thought to be involved in transcriptional activation. Class B and class C HSFs lack a defined aromatic/hydrophobic/acidic (AHA)-type activation domain (reviewed in Miller and Mittler, Annals of Botany, 2006, 98: 279-288). The absence of an activation domain, as well as their inability to rescue the yeast HSF1 mutation, has led to the assumption that class B HSFs function as repressors (Boscheinen et al., Mol. Gen. Genetics, 1997, 255: 322-331; Czarnecka-Vemer et al., Plant Mol. Biol., 2000, 43: 459-471, Czarnecka-Vemer et al., Plant Mol. Biol., 2004, 56: 57-75). However, HSFB1 was recently demonstrated to function as a novel co-regulator of the tomato HSFA1 or HSFA2 enhancing their transcriptional activity (Bharti et al., Plant Cell, 2004, 16: 1521-1535). In tomato (Lycopersicon peruvianum), (i) HSFA1a is the master regulator responsible for heat stress (hs)-induced gene expression including synthesis of HSFA2 and HSFB1. It is indispensable for the development of thermotolerance. (ii) Although functionally equivalent to HSFA1a, HSFA2 is exclusively found after heat shock induction and represents the dominant HSF, the “working horse” of the heat shock response in plants subjected to repeated cycles of heat shock and recovery in a hot summer period. Tomato HSFA2 is tightly integrated into a network of interacting proteins (HSFA1a, Hsp17-CII, Hsp17-CI) influencing its activity and intracellular distribution. (iii) Because of structural peculiarities, HSFB1 acts as co-regulator enhancing the activity of HSFA1a and/or HSFA2. But in addition, it cooperates with yet to be identified other transcription factors in maintaining and/or restoring housekeeping gene expression. (reviewed in Baniwal et al., J. Biosci., 2004, 29: 471-487)
The situation in Arabidopsis seems to be different in several aspects. A single HSF as master regulator could not be identified (Lohmann et al., Mol. Gen. Genomics, 2004, 271: 11-21). In addition, Arabidopsis HSFB1 is not comparable to its tomato counterpart (Kotak et al., Plant J., 2004, 39: 98-112).
Based on the analysis of Arabidopsis HSFs, it is suggested that there is a high degree of specialization in the response of specific HSFs to particular stress conditions. Thus, for example, AtHSFA9 appears to be specific to salt, drought and cold stress, while AtHSFA6a and AtHSFA6b appear to be cold and salt specific. With the exception of AtHSFA2 and AtHSFB1, the pattern of HSF expression during heat stress is different from the pattern of HSF expression during other stresses. Because HSEs are found in the promoters of many defense genes (see, e.g., Rizhsky et al., J. Biol. Chem., 2004, 279: 11736-11743), it is possible that different HSFs, expressed during different stresses, activate or control different defense pathways. The combinatorial function of HSFs could therefore be responsible for stress-specific expression of HSPs or other defense genes, and specific stress conditions could therefore cause activation of a particular set(s) of different HSFs (Rizhsky et al., Plant Physiol., 2004, 134: 1683-1696). Thus, in addition to being potentially redundant, the HSF gene network in plants is highly flexible and specialized. It controls the response of plants to diverse stress conditions, as well as potentially their combination (Rizhsky et al., Plant Physiol., 2004, 134: 1683-1696; Mittler, Trends in Plant Sci., 2006, 11: 15-19). Indeed, functional interdependence studies between HSFs, co-immunoprecipitation and yeast one-hybrid assays suggest that all class A HSFs of tomato can interact with each other, potentially forming hetero-oligomers (Scharf et al., Mol. Cell. Biol., 1998, 18: 2240-2251; Bharti et al., Plant J., 2000, 22: 355-365). Furthermore, different HSFs can associate with each other potentially functioning as co-activators or co-repressors (Baniwal et al., J. Biosciences, 2004, 29: 471-487). Taken together, the complexity of the HSF gene network of plants is evident on at least five different levels: (1) a large number of HSF genes are present in the plant genome; (2) each HSF gene can potentially bind to its own promoter, as well as to the promoters of all other HSF genes; (3) monomers encoded by different HSF genes can interact leading to activation or suppression of transcription; (4) monomers encoded by different HSF genes can interact affecting nuclear targeting and retention; and (5) spatial and temporal expression patterns of HSFs could affect different responses in different tissues. These features make the HSF gene network a highly redundant and specialized network that functions in a stress- or developmental-specific manner (reviewed in Miller and Mittler, Annals of Botany, 2006, 98: 279-288).
Under normal conditions, mammalian HSF1 exists in the cell as an inactive monomer. Following exposure to elevated temperature, HSF1 trimerizes and apparently relocates to the nucleus where it binds to specific sites in HSP promoters upstream of the transcription initiation site (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Westwood, et al., Mol. Cell. Biol. 1993; 13:3481-3486; Westwood, et al., Nature 1991; 353:822-827). Similarly, plant HSF1A undergoes trimerization and DNA binding upon stress (Mishra et al., Genes Dev, 2002, 16: 1555-1567). The HSF binding site contains arrays of inverted repeats of the element NGAAN designated HSE (Heat Shock Element). The same evolutionarily conserved HSE sequence is recognized by all members of the HSF protein family and is universal to all eukaryotic species (Kim et al., Protein Sci. 1994; 3:1040-1051). The heat shock promoter is primed for rapid activation in response to heat shock. Many factors of the basal transcription machinery are bound to the promoter prior to heat shock including GAGA factor, TFIID, transcriptionally arrested RNA polymerase II located 21-35 nucleotides downstream of the transcription start site, and presumably some other transcription factors (Shopland, et al., Genes Dev. 1995; 9:2756-2769). The partitioning of HSF molecules between the nucleus and cytoplasm is a subject to some controversy since, in the case of Xenopus laevis, HSF1 was shown to be a nuclear protein before heat shock (Mercier, et al., J. Biol. Chem. 1997; 272:14147-14151), while in most studies employing mammalian cells, HSF1 was found in both the cytoplasm and nucleus under normal conditions (Sarge, et al., Mol. Cell. Biol. 1993b; 13:1392-1407). Interestingly, heat shock treatment of HeLa cells results in rapid and reversible localization of HSF1 in specific nuclear granules, which constitute a novel type of nuclear protein compartmentalization (Cotto, et al., Journal of Cell Science 1997b; 110:2925-2934). The granules appear within 30 sec of heat shock treatment and rapidly disappear upon shift to normal temperature. However, the functional significance of this phenomenon is still unknown.
The overall structure of HSF1 is conserved among species as distant as Drosophila and human. The DNA-binding domain is just over 100 amino acids long and is situated close to the N-terminus of the molecule. This domain is about 70% homologous between human HSF1 and Drosophila HSF and shows 55% homology between human HSF1 and yeast HSF. The leucine zipper domain, which is C-terminal with respect to the DNA-binding domain, is even more conserved showing 79% homology between human and Drosophila. In vertebrates, this domain comprises three hydrophobic heptad repeats with an additional heptad repeat located in the C-terminus of HSF1. It has been implicated in the maintenance of the inactive monomeric state of HSF1 under non-stressful conditions (Wu, et al., In The Biology of Heat Shock Proteins and Molecular Chaperones, 1994 (New York: Cold Spring Harbor Press), pp. 395-416). It has been suggested that the function of HSFs as transcription activators resides in short activator peptide motifs (AHA motifs) in their C-terminal domains characterized by aromatic (W, F, Y), large hydrophobic (L, I, V) and acidic (E, D) amino acid residues (Treuter et al., Mol. Gen. Genet., 1993, 240: 113-125; Döring et al., Plant Cell, 2000, 12: 265-278; Kotak et al., Plant J., 2004, 39: 98-112). Similar AHA motifs were found and functionally characterized in the centre of many other transcription factors of yeast and mammals, e.g. VP16, RelA, Spl, Fos, Jun, Gal4, Gcn4 as well as the steroid and retinoic acid receptors (see summary and references in Döring et al., Plant Cell, 2000, 12: 265-278; Kotak et al., Plant J., 2004, 39: 98-112).
A number of models have been proposed to explain how HSF activation is regulated, most of them focusing on repression of the inactive monomer under normal conditions as the most probable mode of regulation. Several lines of evidence suggest the existence of a titratable cellular factor that acts to repress HSF under normal conditions by keeping it in a monomeric form. Indeed, overexpression of both HSF1 and HSF2 in 3T3 mouse fibroblasts resulted in constitutive activation of their DNA-binding activity and transcription of HSP genes (Sarge, et al., Mol. Cell. Biol. 1993a; 13:1392-1407). The observed effect could reflect either general cellular stress caused by the drastic increase in HSF concentration or titration of the negative regulator of HSF, which is present in limiting amounts. Furthermore, expression of human HSF1 in Drosophila cells results in a decrease of the activation threshold temperature by 9 degrees, to the temperature characteristic for the heat shock conditions in Drosophila (32° C.) instead of 41° C.—a characteristic threshold for mammalian cells. At the same time, Drosophila HSF expressed in human cells remained constitutively active even when the temperature was lowered to 25° C.—the normal growth temperature for Drosophila (Clos, et al., Nature 1993; 364:252-255). Similarly, Arabidopsis HSF remained active in Drosophila and human cells even under control conditions (Hubel, et al., Mol. Gen. Genet. 1995; 248:136-141). Taken together, these results strongly suggest that the intracellular environment rather than structure of the HSF molecule determines its behavior in response to heat stress. These data are consistent with the possibility that HSF activation is mediated by a specific stimulating factor(s).
The process of HSF1 activation can be divided into at least two steps: 1) trimerization and acquisition of DNA binding activity; 2) acquisition of transactivation competence, which is correlated with hyperphosphorylation of the factor. Treatment with salicylate and other non-steroid anti-inflammatory drugs induces HSF trimerization and DNA binding but fails to stimulate transcription of HSP genes (Jurivich, et al., J. Biol. Chem. 1995a; 270:24489-24495). However, majority of HSF regulation occurs at the level of its trimerization.
The model of HSF regulation, where HSF activity is a subject to the negative feedback mechanism involving inducible HSP72 and other chaperones, has been a paradigm for a decade. According to this model, HSF monomer is present in the complex with HSP72 and other chaperones (most notably HSP90) under normal conditions. Trimerization of HSF molecules is thought to occur spontaneously as soon as negative regulation by HSPs has been relieved. Indeed, in a number of studies HSF has been shown to possess intrinsic responsiveness to heat (Zuo, et al., Mol. Cell. Biol. 1995; 15:4319-4330; Farkas, et al., Molecular and Cellular Biology 1998a; 18:906-918). However, the HSF concentrations used in these studies far exceeded those found in the cell, which questions the physiological relevance of the data. Furthermore, although all these studies imply that HSF trimerization occurs spontaneously once the negative regulation is relieved, the existence of a positive regulation of HSF activity can not be ruled out. For example, the rapid, specific and reversible formation of HSF granules in nuclei during heat shock (Cotto, et al., Journal of Cell Science 1997a; 110:2925-2934; Jolly, et al., Journal of Cell Science 1997; 110:2935-2941) testifies against spontaneous mechanism given the relatively low number of HSF molecules in the cell, their even distribution throughout cytoplasm under normal conditions, and molecular crowding effect due to very high total protein concentration in the cell as compared to in vitro experimental systems.
HSPs, and HSP70 family in particular, is considered a part of a protective mechanism against certain pathological conditions, including ischemic damage, infection, and inflammation (Pockley, Circulation 2002; 105:1012-1017). In the case of inflammation, a protective role of HSPs has been shown in a variety of experimental models (Jattela et al., EMBO J. 1992; 11:3507-3512; Morris et al., Int. Biochem. Cell Biol. 1995; 27:109-122; Ianaro et al., FEBS Lett. 2001; 499:239-244; Van Molle et al., Immunity 2002; 16:685-695). For example, Ianaro et al. (Mol. Pharmacol. 2003; 64:85-93) have recently demonstrated that HSF1-induced (see below) HSP72 expression in the inflamed tissues and activation of the heat shock response is closely associated with the remission of the inflammatory reaction. It follows, that HSP genes may function as anti-inflammatory or “therapeutic” genes, and their selective in vivo transactivation may lead to remission of the inflammatory reaction (Ianaro et al., FEBS Lett. 2001; 499:239-244 and Ianaro et al., FEBS Lett. 2001; 508:61-66).
A potential therapeutic value of causing increased expression of HSPs in individuals suffering from cerebral or cardiac ischemia and neurodegenerative diseases has been also suggested (Klettner, Drug News Perspect. 2004; 17:299-306; Hargitai et al., Biochem. Biophys. Res. Commun. 2003; 307:689-695; Yenari et al., Ann. Neurol. 1998; 44:584-591; Suzuki et al., J. Mol. Cell. Cardiol. 1998; 6:1129-1136; Warrik et al., Nat. Genet. 1999; 23:425-428). For example, Zou et al. (Circulation 2003; 108:3024-3030) have recently shown that cardiomyocyte cell death induced by hydrogen peroxide was prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42° C. for 60 minutes activated HSF1, which played a critical role in survival of cardiomyocytes from oxidative stress. Ischemia/reperfusion injury has been reported to induce apoptosis in cardiomyocytes (Fliss and Gattinger, Circulation 1996; 79:949-956). Zou et al. (Circulation 2003; 108:3024-3030) have also demonstrated that, in the heart of transgenic mice overexpressing a constitutively active form of HSF1 (and having elevated levels of HSPs 27, 70 and 90 in the heart), ischemia followed by reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct size was smaller, and cardiomyocyte death was less than in wild-type mice. These results suggest that elevated activity of HSF1 (and levels of HSPs) exert protective effect on the electrical activity of myocardium against ischemia/reperfusion injury (see also Plumier et al., J. Clin. Invest. 1995; 95:1854-1860; Marber et al., ibid., pp. 1446-1456; Radford et al., Proc. Natl. Acad. Sci. USA, 1996; 93:2339-2342).
HSPs and HSF1 have been also implicated in protection against a variety of neurodegenerative disorders that involve aberrant protein folding and protein damage. Neuronal cells are particularly vulnerable in this sense as HSF activity and HSP expression are relatively weak in such cells and motor neurons appear to require input of HSP secreted from adjacent glial cells to maintain adequate molecular chaperone levels (Batulan et al., J. Neuosci. 2003; 23:5789-5798; Guzhova et al., Brain Res. 2001; 914:66-73).
Polyglutamine (polyQ) expansion is a major cause of inherited neurodegenerative diseases called polyglutamine diseases. Several polyQ diseases have been identified, including Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and five forms of spinocerebellar ataxia. Aggregates or inclusion bodies of polyQ proteins (e.g., huntingtin) within the nucleus, or in the cytoplasm of neuronal cells in some Huntington's disease patients, are a prominent pathological hallmark of most polyQ diseases (Davies et al., Cell 1997; 90:537-548; DiFiglia et al., Science 1997; 277:1990-1993). Formation of polyQ protein inclusions correlates with an increased susceptibility to cell death (Warrik et al., Cell 1998; 94:939-49). The early stages of aggregates are highly toxic to cells (Bucciantini et al., Nature 2002; 416:507-11). Suppression of aggregates should be beneficial to cells and could delay the progression of polyQ diseases (Sanchez et al., Nature 2003; 421:373-9). HSPs have been implicated in many of these neurodegenerative diseases based on the association of chaperones with intracellular aggregates. For example, live cell imaging experiments show that Hsp70 associates transiently with huntingtin aggregates, with association-dissociation properties identical to chaperone interactions with unfolded polypeptides (Kim et al., Nat. Cell Biol. 2002; 4:826-31). This suggests that these chaperone interactions may reflect the efforts of Hsp70 to direct the unfolding and dissociation of substrates from the aggregate. Moreover, overexpression of the Hsp70 chaperone network suppresses aggregate formation and/or cellular toxicity. A critical protective role for small HSPs (HSP27) has been also recently demonstrated in Huntington's disease (Wyttenbach et al., Human Mol. Gen. 2002; 11:1137-51). Collectively, these observations have led to the hypothesis that the elevated levels of heat shock proteins reduce or dampen aggregate formation and cellular degeneration (Warrick et al, Nat. Genet. 1999; 23:425-8; Krobitsch and Lindquist, Proc. Natl. Acad. Sci. USA 2000; 97:1589-94). Importantly, HSF1 overexpression suppressed polyQ-inclusion formation even better than any combination of HSPs in culture cells and in Huntington's disease model mice extending their life span (Fujimoto et al., J. Biol. Chem. 2005; 280:34908-16).
Multiple HSPs are also overexpressed in brains from Alzheimer's (AD) and Parkinson's disease (PD) patients and found to be associated with senile plaques and Lewy bodies, respectively. These HSPs may be involved in neuroprotection by various mechanisms ranging from microglia activation and clearance of amyloid-β peptides to suppression of apoptosis (Kitamura and Nomura, Pharmacol Ther. 2003; 97:35-33).
Aging is also associated with the decrease in activity of HSF (Tonkis and Calderlwood, Int. J. Hyperthermia 2005; 21:433-444). Indeed, neurodegenerative diseases often occur later in life when heat shock genes seem to be induced poorly (Soti and Csermely, Exp. Gerontol. 2003; 38:1037-40; Shamovsky and Gershon, Mech. Ageing Dev. 2004; 125:767-75). Moreover, it has been recently shown that induction of heat shock either by temperature or HSF overexpression could extend life span in model organisms. For example, the heat shock response has recently been implicated in the regulation of longevity in C. elegans in a pathway that overlaps with the insulin signaling pathway (Hsu et al., Science 2003; 300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64). Reduction of HSF1 levels cause a decreased life span in C. elegans, similar to life span effects observed in mutants of Daf-16, a FOXO transcription factor in the insulin signaling pathway. Daf-16 and HSF1 share a subset of downstream target genes, including small HSPs. RNA interference experiments showed that a decrease in small HSPs and other HSPs leads to a decrease in longevity (Hsu et al., Science 2003; 300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64). Similarly, Walker et al. (Aging Cell 2003; 2:131) have demonstrated that overexpression of HSP16 can extend C. elegans' life span. Therefore, in addition to the prevention of diseases of aging, increased levels of HSPs may lead to increases in life span (Westerheide and Morimoto, J. Biol. Chem. 2005, 280:33097-100).
Heat shock is a known transcriptional activator of human immunodeficiency virus type 1 (HIV) long terminal repeat (LTR). However, HIV LTR suppression can occur under hyperthermic conditions. Specifically, suppression of the HIV LTR was observed in a conditional expression system for gene therapy applications that utilizes the heat-inducible promoter of the human heat shock protein 70B gene to enhance the HIV LTR-driven therapeutic gene expression after hyperthermia (temperature higher than 37° C.) (Gerner et al., Int. J. Hyperthermia 2000; 16:171-181). Similarly, the inhibition of HIV transcription has been reported after whole-body hyperthermia at 42° C. in persons with AIDS (Steinhart et al., J. AIDS Hum. Retrovirol. 1996; 11:271-281). Recently demonstrated ability of a mutant transcriptionally active HSF1 (lacking C-terminal residues 203-315) to suppress HIV promoter activity further suggests that HSF1 could serve as a tool for the treatment of AIDS (Ignatenko and Gerner, Exp. Cell Res. 2003; 288:1-8; see also Brenner and Wainberg, Expert Opin. Biol. Ther. 2001; 1:67-77).
Due to interaction of HSPs with numerous regulatory proteins (e.g., NF-κB, p53, v-Src, Raf1, Akt, steroid hormone receptors) and pathways (e.g., inhibition of c-Jun NH2-terminal kinase (JNK) activation, prevention of cytochrome c release, regulation of the apoptosome, prevention of lysosomal membrane permeabilization, prevention of caspase activation) involved in the control of cell growth, the increased production of HSPs has potent anti-apoptotic effect (Bold, et al., Surgical Oncology-Oxford 1997; 6:133-142; Jaattela, et al., Exp. Cell Res. 1999; 248:30-43; Nylandsted, et al., Ann. N. Y. Acad. Sci. 2000; 926:122-125; Pratt and Toft, Exp. Biol. Med. (Maywood) 2003; 228:111-33; Mosser and Morimoto, Oncogene 2004; 23:2907-18). Anti-apoptotic and cytoprotective activities of HSPs directly implicate them in oncogenesis (Jolly and Morimoto, J. Natl. Cancer Inst. 2000; 92:1564-72; Westerheide and Morimoto, J. Biol. Chem. 2005, 280:33097-100). Many cancer cells display deregulated expression of HSPs, whose elevated levels contribute to the resistance of cancerous cells to chemo- and radiotherapy. Different subfamilies of HSPs including HSP70, HSP90, and HSP27 were found to be expressed at abnormal levels in various human tumors (Cardoso, et al., Ann. Oncol. 2001; 12:615-620; Kiang, et al., Mol. Cell Biochem. 2000; 204:169-178). In some cases, HSPs are expressed at cell surface in tumors, most probably serving as antigen presenting molecules in this case (Conroy, et al., Eur. J. Cancer 1998; 34:942-943). Both HSP70 and HSP90 were demonstrated to mediate cytoplasmic sequestration of p53 in cancer cells (Elledge, et al., Cancer Res. 1994; 54:3752-3757). Inactivation of wild-type p53 function has been observed in variety of cancer cells and is in fact one of the most common hallmarks in human cancer (Malkin, et al., J. Neurooncol. 2001; 51:231-243). Other studies have demonstrated that HSP70 has a potent general antiapoptotic effect protecting cells from heat shock, tumor necrosis factor, serum starvation, oxidative stress, chemotherapeutic agents (e.g., gemcitabine, torootecan, actinomycin-D, campothecin, and etoposide), and radiation (Jaattela, et al., EMBO J. 1992; 11:3507-3512; Jaattela, et al., J. Exp. Med. 1993; 177:231-236; Simon, et al., J. Clin. Invest 1995; 95:926-933; Karlseder, et al., Biochem. Biophys. Res. Commun. 1996; 220:153-159; Samali and Cotter, Exp. Cell Res. 1996; 223:163-170; Sliutz et al., Br. J. Cancer 1996; 74:172-177). At the same time, HSP70 is abundantly expressed in human malignant tumors of various origins, not only enhancing spontaneous growth of tumors, but also rendering them resistant to host defense mechanisms and therapeutic treatment (Ciocca, et al., Cancer Res. 1992; 52:3648-3654). Therefore, finding a way to suppress HSP overproduction in cancerous cells will be invaluable for increasing the efficacy of the existing anti-cancer therapeutic approaches.
HSF1-mediated induction of HSPs has been also implicated in protection of sensory hair cells against acoustic overexposure, hyperthermia and ototoxic drugs. It has been shown that mice lacking HSF1 have reduced recovery from noise-induced hearing loss (Altschuler et al., Audiol Neotol. 2003; 7:152-156). Similarly, Sugahara et al. (Hear Res. 2003; 182:88-96) have demonstrated that the loss of sensory hair cells was more significant in HSF1-null mice compared with that of wild-type mice when mice were subjected to acoustic overexposure. They have also shown that the loss of both the sensory hair cells and the auditory function induced by acoustic overexposure was inhibited by pretreatment of the inner ear with local heat shock.
Developing stress tolerant plants has been a growing agricultural concern as will help farmers across the world to cope with ever-present environmental stresses and with increased stresses due to climate change and will also help to achieve higher yields. However, traditional plant breeding strategies to develop new lines of plants that exhibit resistance (tolerance) to various types of biotic and abiotic stresses are relatively slow and require specific resistant lines for crossing with the desired line. Limited germplasm resources for stress tolerance and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Additionally, the cellular processes leading to stress tolerance (e.g., drought, cold, and salt tolerance) in plants are complex in nature and involve multiple mechanisms of cellular adaptation and numerous metabolic pathways. This multi-component nature of stress tolerance has not only made breeding for stress tolerance largely unsuccessful, but has also limited the ability to genetically engineer stress tolerant plants using biotechnological methods. Thus, generation of genetically engineered plants where multiple stress-response pathways are affected appears an attractive alternative approach.
Based on the information provided above, HSF appears to be an attractive therapeutic target for regulating HSP synthesis to combat various diseases in animals and to generate stress-resistant plants (Mestril et al., J. Clin. Invest. 1994; 93:759-67; Morimoto, et al., Genes Dev. 1998; 12:3788-3796; Jolly and Morimoto, J. Natl. Cancer Inst. 2000; 92:1564-72; Ianaro et al., FEBS Lett. 2001; 499:239-44; Calderwood and Asea, Int. J. Hyperthermia 2002; 18:597-608; Zou et al., Circulation 2003; 108:3024-30; Westerheide and Morimoto, J. Biol. Chem. 2005; 280:33097-100).